title: Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects.
abstract: The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.
authors.affiliation: Center for Radiological Research, Columbia University Medical Center, New York, New York 10032, USA. ht2231@columbia.edu
authors.forename: Helen C
authors.initials: HC
authors.lastname: Turner
authors.forename: David J
authors.initials: DJ
authors.lastname: Brenner
authors.forename: Youhua
authors.initials: Y
authors.lastname: Chen
authors.forename: Antonella
authors.initials: A
authors.lastname: Bertucci
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.forename: Hongliang
authors.initials: H
authors.lastname: Wang
authors.forename: Oleksandra V
authors.initials: OV
authors.lastname: Lyulko
authors.forename: Yanping
authors.initials: Y
authors.lastname: Xu
authors.forename: Igor
authors.initials: I
authors.lastname: Shuryak
authors.forename: Julia
authors.initials: J
authors.lastname: Schaefer
authors.forename: Nabil
authors.initials: N
authors.lastname: Simaan
authors.forename: Gerhard
authors.initials: G
authors.lastname: Randers-Pehrson
authors.forename: Y Lawrence
authors.initials: YL
authors.lastname: Yao
authors.forename: Sally A
authors.initials: SA
authors.lastname: Amundson
authors.forename: Guy
authors.initials: G
authors.lastname: Garty
mesh_terms: D000328:Adult; D048430:Cell Shape; D048429:Cell Size; D005720:Gamma Rays; D006493:Heparin / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D011786:Quality Control; D011829:Radiation Dosage; D011874:Radiometry / Q000295:instrumentation* / Q000592:standards; D015203:Reproducibility of Results; D012371:Robotics
chemical_list: C468783:H2AX protein, human; D006657:Histones; D006493:Heparin